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We report here the annotated draft genome sequence of the rhizobium strain BR 2003. This strain is able to establish symbiosis and to fix nitrogen with a broad range of leguminous species. The estimation of the average nucleotide identity confirmed the strain as a member of Bradyrhizobium elkanii.

Made available in DSpace on 2018-12-11T17:10:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The genus Bradyrhizobium comprises bacteria with the ability to form nitrogen-fixing symbioses with legumes. They are of great interest in agriculture, as well as for the production of biopolymers such as polyhydroxyalkanoates. Here, we report the draft genome assembly of Bradyrhizobium elkanii TnphoA 33 comprising 9 Mb, 1,124 contigs, and 9,418 open reading frames. Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias UNESP-Universidade Estadual Paulista University Medical Center Utrecht Departamento de Biologia Aplicada à Agropecuária Faculdade de Ciências Agrárias e Veterinárias UNESP- Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Tecnologia Laboratorio Multiusuario de Sequenciamento em Larga Escala e Expressao Genica UNESP-Universidade Estadual Paulista Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias UNESP-Universidade Estadual Paulista Departamento de Biologia Aplicada à Agropecuária Faculdade de Ciências Agrárias e Veterinárias UNESP- Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Tecnologia Laboratorio Multiusuario de Sequenciamento em Larga Escala e Expressao Genica UNESP-Universidade Estadual Paulista

We investigated the relationship between the genetic diversity of indigenous soybean-nodulating bradyrhizobia and their geographical distribution in the United States using nine soil isolates from eight states. The bradyrhizobia were inoculated on three soybean Rj genotypes (non- Rj , Rj 2 Rj 3 , and Rj 4 ). We analyzed their genetic diversity and community structure by means of restriction fragment length polymorphisms of PCR amplicons to target the 16S-23S rRNA gene internal transcribed spacer region, using 11 USDA Bradyrhizobium strains as reference strains. We also performed diversity analysis, multidimensional scaling analysis based on the Bray-Curtis index, and polar ordination analysis to describe the structure and geographical distribution of the soybean-nodulating bradyrhizobial community. The major clusters were Bradyrhizobium japonicum Bj123, in the northern United States, and Bradyrhizobium elkanii , in the middle to southern regions. Dominance of bradyrhizobia in a community was generally larger for the cluster belonging to B. elkanii than for the cluster belonging to B. japonicum . The indigenous American soybean-nodulating bradyrhizobial community structure was strongly correlated with latitude. Our results suggest that this community varies geographically.

We report here the draft genome sequences of Bradyrhizobium elkanii strains BLY3-8 and BLY6-1, which are incompatible with Rj 3 genotype soybean cultivars. The genome sequences of these strains will be useful to identify a causal gene for this incompatibility.

As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium , including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii , Bradyrhizobium elkanii , B. japonicum , and B. yuanmingense , demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean ( Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N 2 -fixation genes (PCR-RFLP and sequencing of nodY-nodA , nodC , and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum . Another one (CPAC 402) was identified as an indigenous Sinorhizobium ( Ensifer ) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.

SEMIA 690 T is a nitrogen-fixing symbiont of Centrosema pubescens , and comprises the recently described species Bradyrhizobium viridifuturi . Its draft genome indicates that it belongs to the Bradyrhizobium elkanii superclade. SEMIA 690 T carries two copies of the regulatory nodD gene, and the nod and nif operons resemble those of Bradyrhizobium diazoefficiens .

Bradyrhizobium pachyrhizi PAC48 T has been isolated from a jicama nodule in Costa Rica. The draft genome indicates high similarity with that of Bradyrhizobium elkanii . Several coding sequences (CDSs) of the stress response might help in survival in the tropics. PAC48 T carries nodD1 and nodK , similar to Bradyrhizobium ( Parasponia ) ANU 289 and a particular nodD2 gene.

The nodulation of Erythrophleum fordii has been recorded recently, but its microsymbionts have never been studied. To investigate the diversity and biogeography of rhizobia associated with this leguminous evergreen tree, root nodules were collected from the southern subtropical region of China. A total of 166 bacterial isolates were obtained from the nodules and characterized. In a PCR-based restriction fragment length polymorphism (RFLP) analysis of ribosomal intergenic sequences, the isolates were classified into 22 types within the genus Bradyrhizobium . Sequence analysis of 16S rRNA, ribosomal intergenic spacer (IGS), and the housekeeping genes recA and glnII classified the isolates into four groups: the Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi groups, comprising the dominant symbionts, Bradyrhizobium yuanmingense , and an unclassified group comprising the minor symbionts. The nodC and nifH phylogenetic trees defined five or six lineages among the isolates, which was largely consistent with the definition of genomic species. The phylogenetic results and evolutionary analysis demonstrated that mutation and vertical transmission of genes were the principal processes for the divergent evolution of Bradyrhizobium species associated with E. fordii , while lateral transfer and recombination of housekeeping and symbiotic genes were rare. The distribution of the dominant rhizobial populations was affected by soil pH and effective phosphorus. This is the first report to characterize E. fordii rhizobia.

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii . The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA . A large-deletion mutant of B. elkanii , USDA94Δ rtx ::Ω1, which lacks rtxA , ORF1 ( rtxC ), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94Δ rtx ::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.

Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes ( Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis , and Desmodium axillare ) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes ( Platypodium and Machaerium ) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii .

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum . These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum .

Rhizobitoxine is synthesized by the legume symbiont Bradyrhizobium elkanii and the plant pathogen Burkholderia andropogonis . Rhizobitoxine competitively inhibited 1-aminocyclopropane-1-carboxylate (ACC) synthase bLE-ACS2 from the tomato, a key enzyme in the pathway of ethylene biosynthesis. Based on this inhibition of ACC synthase, we have developed a new assay for rhizobitoxine.

We disrupted the rtxC gene on the chromosome of Bradyrhizobium elkanii USDA94 by insertion of a nonpolar aph cartridge. The rtxC mutant, designated Δ rtxC , produced serinol and dihydrorhizobitoxine but no rhizobitoxine, both in culture and in planta. The introduction of cosmids harboring the rtxC gene into the Δ rtxC mutant complemented rhizobitoxine production, suggesting that rtxC is involved in the final step of rhizobitoxine biosynthesis in B. elkanii USDA94. Glycine max cv. Lee inoculated with Δ rtxC or with a null mutant, Δ rtx ::Ω1, showed no foliar chlorosis, whereas the wild-type strain USDA94 caused severe foliar chlorosis. The two mutants showed significantly less nodulation competitiveness than the wild-type strain on Macroptilium atropurpureum . These results indicate that dihydrorhizobitoxine, the immediate precursor of the oxidative form of rhizobitoxine, has no distinct effect on nodulation phenotype in these legumes. Thus, desaturation of dihydrorhizobitoxine by rtxC -encoded protein is essential for the bacterium to show rhizobitoxine phenotypes in planta. In addition, complementation analysis of rtxC by cosmids differing in rtxC transcription levels suggested that rhizobitoxine production correlates with the amount of rtxC transcript.

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In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii . Rhizobium sp. ( Sesbania ) strain IRBG74 was located in the Rhizobium - Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter ). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. ( Chamaecytisus ) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana , Desmodium glutinosum , and Amphicarpaea bracteata , three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata . Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii .